Antimicrobial Potency of Single and Combined Mupirocin and Monoterpenes, Thymol, Menthol and 1,8-Cineole Against Staphylococcus Aureus Planktonic and Biofilm Growth
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The Journal of Antibiotics (2016) 69, 689–696 & 2016 Japan Antibiotics Research Association All rights reserved 0021-8820/16 www.nature.com/ja ORIGINAL ARTICLE Antimicrobial potency of single and combined mupirocin and monoterpenes, thymol, menthol and 1,8-cineole against Staphylococcus aureus planktonic and biofilm growth Domagoj Kifer, Vedran Mužinić and Maja Šegvić Klarić Staphylococcus aureus is one of the most commonly isolated microbes in chronic rhinosinusitis (CRS) that can be complicated due to the formation of a staphylococcal biofilm. In this study, we investigated antimicrobial efficacy of single mupirocin and three types of monoterpenes (thymol, menthol and 1,8-cineole) as well as mupirocin–monoterpene combinations against S. aureus ATCC 29213 and 5 methicilin-resistant S. aureus strains (MRSA) grown in planktonic and biofilm form. MIC against planktonic bacteria as well as minimum biofilm-eliminating concentrations (MBECs) and minimum biofilm inhibitory concentrations (MBICs) were determined by TTC and MTT reduction assay, respectively. The MICs of mupirocin (0.125–0.156 μgml− 1) were three orders of magnitude lower than the MICs of monoterpenes, which were as follows: thymol (0.250–0.375 mg ml − 1) 4 menthol (1 mg ml − 1) 4 1,8-cineole (4–8mgml− 1). Mupirocin-monoterpene combinations showed indifferent effect as compared with MICs of single substances. Mupirocin (0.016–2mgml− 1) failed to destroy the biofilm. The MBECs of thymol and menthol were two- to sixfold higher than their MICs, while 1,8-cineole exerted a weak antibiofilm effect with MBECs 16- to 64-fold higher than MICs. Mixture of mupirocin and 1,8 cineole exerted a potentiated biofilm-eliminating effect, mupirocin–menthol showed antagonism, while effect of thymol–mupirocin mixture was inconclusive. MBICs of antimicrobials were close to their MICs, except 1,8-cineole, MBIC was about three- to fivefold higher. Dominant synergy was observed for mixtures of mupirocin and menthol or thymol, whereas mupirocin-1,8-cineol exerted an indifferent or additive biofilm inhibitory effect. Particular combinations of mupirocin and the monoterpenes could be applied in CRS therapy in order to eliminate or prevent bacterial biofilm growth. The Journal of Antibiotics (2016) 69, 689–696; doi:10.1038/ja.2016.10; published online 17 February 2016 INTRODUCTION treatment of S. aureus-associated wound infections. It is a structural Staphylococcus aureus is one of the most important human colonizers analog of isoleucyl-adenylate (Ile-AMP), which competes with and pathogens that causes a variety of diseases, from mild skin and Ile-AMP for binding sites of the isoleucyl-tRNA synthetases leading soft tissue infections to more serious conditions including pneumonia, to abrogation of protein synthesis in bacteria.9,10 Recently, Bode endocarditis, osteomyelitis and sepsis.1 Also, it is one of the most et al.11 showed that the nasal decolonization of S. aureus carriers by commonly isolated microbes in chronic rhinosinusitis (CRS), a mupirocin significantly reduces the risk of S. aureus infections after chronic disease of the paranasal sinuses that affects a large number surgery. However, data on the efficacy of mupirocin treatment in of individuals in the developed world.2–4 Apart from the methicilin biofilm-associated nasal S. aureus infections as well as mupirocin resistance of S. aureus (MRSA), infections with S. aureus can even antibiofilm activity in vitro are scarce. The development of antibiotic further complicate owing to biofilm formation. In CRS, mucosal resistance in pathogenic bacteria increased general interest in studying changes result in favorable conditions for the development of a the antimicrobial potency of phytochemicals including monoterpenes S. aureus biofilm. Once a biofilm is established, its resistance to both menthol, thymol and 1,8-cineole. These amphipathic phytochemicals host defences and antimicrobials (restricted penetration, decrease in with prevalent hydrophobicity evoke alterations of S. aureus bacterial metabolism and growth rate, increase in antibiotic-degrading membrane permeability causing leakage of intracellular material.12 enzymes accumulation and enhancement of exchanging rate of genes The studies undertaken so far have shown that some phytochemicals encoding for resistance) leads to chronic inflammation.5–8 Mupirocin including thymol might have impact on bacterial biofilm formation or pseudomonic acid A is natural antibiotic produced by Pseudomonas and development, acting as control agents of bacterial adhesion and fluorescens, which has been successfully used as topical agent in the cell-to-cell communication known as quorum sensing-QS.12 However, Department of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia Correspondence: Professor MŠ Klarić, Department of Microbiology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Schrottova 39, 10000 Zagreb, Croatia. E-mail: [email protected] Received 17 October 2015; revised 16 January 2016; accepted 22 January 2016; published online 17 February 2016 Mupirocin and monoterpenes against Staphylococci DKiferet al 690 the biofilm eradication ability of such phytochemicals is insufficiently Biofilm formation investigated. Taking into account the amphipathic nature of menthol, Biofilm was formed according to the method by Walencka et al.15 with thymol and 1,8-cineole, these monoterpenes could penetrate through minor modifications. Stock inoculum was prepared as described previously − the exopolysaccharide matrix of S. aureus biofilm and enhance the andadjustedto1,5×106 c.f.u. ml 1 in Tryptic soy broth (TSB, Difco, Le Pont activity of mupirocin trough membrane permeability disturbances. de Claix, France), supplemented with 0,25% D-(+)-glucose (Kemika, Zagreb, Therefore, the aim of our study was to investigate the antimicrobial Croatia; TSBGlc). Diluted bacterial suspensions were incubated for 24 h at 37 ° C, aerobically. efficacy of mupirocin alone and the mentioned monoterpenes as well – Following incubation, the suspension was diluted 20-fold with TSBGlc. as mupirocin monoterpene combinations against S. aureus planktonic μ fi A total of 100 l of this suspension was added to each well of 96-well tissue and bio lm modes of growth in vitro. culture plate (Nuncoln Surface, Nunc, Thermo Fisher Scientific, Waltham, MA, USA) and incubated for 24 h at 37 °C aerobically, to form the biofilm. To MATERIALS AND METHODS visualize a biofilm, a method by Kairo et al.16 was used, with a few Bacterial strains modifications. After biofilm formation, wells were emptied and gently rinsed Staphylococcus aureus ATCC 29213 and clinical MRSA isolates (n = 5) were with 100 μl of saline, to remove planktonic bacteria and preserve the biofilm. used in this study. All of the strains (MFBF 10674, MFBF 10676, MFBF 10677, After rinsing, 50 μl of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- MFBF 10679, MFBF 10680) were taken from the microbe culture collection lium bromide, MTT reagent, Sigma) was added into each well and incubated of Department of Microbiology, Faculty of Pharmacy and Biochemistry, for 2 h at 37 °C. MTT is metabolized by living bacterial cells forming purple University of Zagreb. formazan crystals. The formazan crystals were dissolved by adding 150 μlof dimethyl sulfoxide (DMSO, Sigma). The plate was shaken for 10 min until all Antimicrobials formazan crystals dissolved. The absorbance was measured on a microplate Mupirocin calcium dihydrate was donated by Pliva (Pliva, Zagreb, Croatia), reader (iEMS, Labsystems) at wavelength of 540 nm. whereas monoterpenes L-menthol(Sigma,StLouis,MO,USA),thymol(Kemika, Zagreb, Croatia) and 1,8-cineole (Merck, Darmstadt, Germany) were purchased. Determination of minimum biofilm-eliminating concentration Immediately before the assay, mupirocin calcium dihydrate was dissolved The biofilm of each S. aureus strain was grown on the bottom of wells of a and diluted in an appropriate broth. Monoterpenes were dissolved in 100% 96-well microtiter plate as described above. Initial biofilm was stained and its ethanol (Kemika, Zagreb, Croatia) and diluted in an appropriate broth. Stock absorbance was measured as described above. The obtained absorbance was solution of Tween 80 in sterile water (5%) was used for homogenization of defined as the absorbance of the initial biofilm, Aib. monoterpene alcoholic solutions. In other wells, the intact initial biofilm was treated by adding 100 μlof Determination of MIC against planktonic bacteria antimicrobial agent solution. Concentration ranges of antimicrobials − − − A stock culture of S. aureus strains was prepared, grown at 37 °C aerobically for were 0.016 μgml 1–2mgml 1 (mupirocin), 0.5–64 mg ml 1 (menthol), − − 24 h in Müller–Hinton broth (Merck, Germany). A 1.5 × 108 colony-forming 0.25–32 mg ml 1 (thymol) and 2–256 mg ml 1 (1,8-cineole). Concentrations unit (c.f.u.) per ml inoculum was prepared using a nephelometer (BioMereiux, were prepared by diluting stock solutions with Tryptic soy broth (TSB, Merck, Marcy-l'Etoile, France) and adjusted to a final concentration 5 × 105 c.f.u. ml − 1 Germany). Control strains were grown in broth with and without 5–10% of in Müller–Hinton broth containing antimicrobials. The determination of MICs ethanol + tween. Plates were incubated for 24 h at 37 °C aerobically. Following for mupirocin, menthol, thymol and 1,8-cineole were carried out using a incubation, the wells were emptied and rinsed, and the biofilm remaining on twofold microdilution method, according to Clinical and Laboratory Standards the bottom of the wells was stained and measured as described previously. The 13 Institute guidelines. Concentrations of twofold serial-diluted solutions in obtained absorbance was defined as the absorbance of surviving biofilm, Asb. Müller–Hinton broth used for determining MIC for mupirocin were in a Antimicrobial treatment of each strain as well as controls was derived in − 1 range from 0.0625 to 128 μgml , concentrations of menthol, thymol and quadruplicate. Viability was calculated using Aib and Asb by Equation (2). 1,8-cineole were in ranges of 0.03125–64 mg ml − 1,0.125–128 mg ml − 1 and (Minimum biofilm-eliminating concentration (MBEC) is defined as minimum 0.125–128 mg ml − 1, respectively.